This invention relates to an assay method for determining hydrolytic enzyme activity and to polypeptides and modified polypeptides useful therein. In particular, it relates to a method for determining hydrolytic enzyme activity which is applicable to the rapid screening of large numbers of potential enzyme inhibitors or stimulators.
The availability of methods for the evaluation of hydrolytic enzymes not only permits the study of the enzyme itself, for example its activity against modified substrates or its kinetics, but also provides for evaluating substances as inhibitors or stimulators for enzymes. Methods for the rapid screening of hydrolytic enzyme inhibitors and stimulators exist where the recognition sites for the enzyme are on one side of the cleavage site of the substrate. For example, in a tetrapeptide R1 ? ##STR1## wherein X is a marking element e.g. a fluorescent marker, and A, B, C, and D represent amino acid residues, any protease that cleaves at the C-terminal side of residue D with recognition sites in the residues N-terminal to D will release the marker X which, when released, will exhibit a different absorption spectrum and can be readily detected in the visible spectrum. The intensity of the absorption will correspond to the extent cleavage has occurred. Accordingly one can screen for inhibitors of such a protease by simply adding the inhibitor to the protease reaction mixture and directly measuring the extent of cleavage. Numerous potential inhibitors thus can be screened rapidly. When, however, the protease requires recognition sites on both sides of the cleavage site such as shown below ##STR2## wherein A-I are amino acid residues and X is the marker, then X will not be released, as described above, to provide the requisite absorption change to measure the extent of cleavage. Thus, the extent to which cleavage occurred has to be determined indirectly, e.g. by chromatographic analysis such as with HPLC. Accordingly, prior to the present invention there was no adequate method available for rapidly screening hydrolytic enzyme inhibitors or stimulators where the enzyme requires recognition elements on both sides of the cleavage site of the substrate.
The method of this invention is applicable to hydrolytic enzymes in general and can be used, for example, in assaying for proteases, glycosidases and nucleases.